Neal D. Epstein

Mutations in either the essential or regulatory light chains of myosin are associated with a rare myopathy in human heart and skeletal muscle

The muscle myosins are hexomeric proteins consisting of two heavy chains and two pairs of light chains, the latter called essential (ELC) and regulatory (RLC). The light chains stabilize the long alpha helical neck of the myosin head. Their function in striated muscle, however, is only partially understood. We report here the identification of distinct missense mutations in a skeletal/ventricular ELC and RLC, each of which are associated with a rare variant of cardiac hypertrophy as well as abnormal skeletal muscle.We show that myosin containing the mutant ELC has abnormal function, map the mutant residues on the three–dimensional structure of myosin and suggest that the mutations disrupt the stretch activation response of the cardiac papillary muscles.

Long-term results of dual-chamber (DDD) pacing in obstructive hypertrophic cardiomyopathy. Evidence for progressive symptomatic and hemodynamic improvement and reduction of left ventricular hypertrophy.

BackgroundWe previously reported that 6 to 12 weeks of dual-chamber (DDD) pacing results in clinical and hemodynamic improvement in obstructive hypertrophic cardiomyopathy (HCM). This study examines the long-term results of DDD pacing in obstructive HCM. Methods and ResultsDDD devices were implanted in 84 patients (mean age, 49 ± 16 years) with obstructive HCM and severe drug-refractory symptoms. At a mean follow-up of 2.3 ± 0.8 years (maximum, 3.5 years), the New York Heart Association (NYHA) functional class had improved significantly (1.6 ± 0.6 versus 3.2 ± 0.5, P < .00001). Symptoms were eliminated in 28 patients (33%), improved in 47 patients (56%), but remained unchanged in 7 patients (8%). Two patients died suddenly (97% cumulative 3-year survival rate). In 74 patients with significant left ventricular outflow tract (LVOT) obstruction at rest, the LVOT gradients were significantly reduced at follow-up (27 ± 31 versus 96 ± 41 mm Hg, P < .00001). Symptoms and provokable LVOT gradients were also reduced in all 10 patients without significant resting but with provokable LVOT obstruction. Persistence of the LVOT obstruction and symptoms was attributed to inability to pre-excite the interventricular septum (n = 8) and onset of atrial fibrillation (n = 7). Fifty patients had two cardiac catheterization evaluations, 3 ± 1 and 16 ± 4 months after implantation of a pacemaker. In this subgroup, the NYHA functional class improved from 3.2 ± 0.5 at baseline to 1.8 ± 0.7 at the initial evaluation (P < .00001), but with a further significant improvement at the second evaluation: 1.4 ± 0.6, P < .001. This symptomatic improvement was associated with progressive reduction of LVOT gradient at the two evaluations: baseline, 100 ± 47 mm Hg; first evaluation, 41 ± 36 mm Hg (P < .0001); and second evaluation, 29 ± 34 mm Hg (P < .01). Despite the presence of left bundle branch block, DDD pacing reduced LVOT obstruction significantly in 15 patients (LVOT gradient, baseline 89 ± 36 mm Hg versus 18 ± 26 mm Hg at follow-up, P < .0001). There was a weak but significant correlation between the reduction in LVOT gradients accomplished by AV pacing before implantation of DDD device and the eventual reduction in LVOT gradients recorded at the follow-up evaluation (r = .38, P = .0017). Echocardiography demonstrated significant thinning of the anterior septum and distal anterior LV wall in the absence of deterioration of LV systolic function. Conclusions(1) Although most of the improvement of symptoms and hemodynamic indexes occurs during the first few months of DDD pacing, further changes are often observed a year later; (2) DDD pacing is associated with an excellent prognosis in a subgroup of severely disabled patients, many of whom present with syncope or presyncope; (3) baseline pacing studies are not essential to identify patients who may benefit from pacing; (4) preexisting left bundle branch block is compatible with severe LVOT obstruction, and DDD pacing is also beneficial in this subgroup; (5) DDD pacing reduces both resting and provokable LVOT obstruction; (6) additional therapy, for example, radiofrequency ablation of the AV node, may be necessary in some patients either to preexcite the interventricular septum or to control atrial fibrillation; and (7) although LV hypertrophy has been considered a primary feature of HCM, pacing appears to reverse LV wall thickness in a significant subset of adult HCM patients.

The Overall Pattern of Cardiac Contraction Depends on a Spatial Gradient of Myosin Regulatory Light Chain Phosphorylation

Evolution of the human heart has incorporated a variety of successful strategies for motion used throughout the animal kingdom. One such strategy is to add the efficiency of torsion to compression so that blood is wrung, as well as pumped, out of the heart. Models of cardiac torsion have assumed uniform contractile properties of muscle fibers throughout the heart. Here, we show how a spatial gradient of myosin light chain phosphorylation across the heart facilitates torsion by inversely altering tension production and the stretch activation response. To demonstrate the importance of cardiac light chain phosphorylation, we cloned a myosin light chain kinase from a human heart and have identified a gain-in-function mutation in two individuals with cardiac hypertrophy.

Skeletal muscle expression and abnormal function of beta-myosin in hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy is an important inherited disease. The phenotype has been linked, in some kindreds, to the beta-myosin heavy chain (beta-MHC) gene. Missense and silent mutations in the beta-MHC gene were used as markers to demonstrate the expression of mutant and normal cardiac beta-MHC gene message in skeletal muscle of hypertrophic cardiomyopathy patients. Mutant beta-myosin, also shown to be present in skeletal muscle by Western blot analysis, translocated actin filaments slower than normal controls in an in vitro motility assay. Thus, single amino acid changes in beta-myosin result in abnormal actomyosin interactions, confirming the primary role of missense mutations in beta-MHC gene in the etiology of hypertrophic cardiomyopathy.

Differences in clinical expression of hypertrophic cardiomyopathy associated with two distinct mutations in the beta-myosin heavy chain gene. A 908Leu----Val mutation and a 403Arg----Gln mutation.

BackgroundThe disease gene for hypertrophic cardiomyopathy (HCM) has been identified as the β-myosin heavy chain (β-MHC) gene in some HCM families. We describe extensive clinical evaluations in two kindreds with two distinct point mutations in the β-MHC gene. Methods and ResultWe used single-strand confirmation polymorphism (SSCP) gel analysis of polymerase chain reaction-amplified products capturing each of the 40 β-MHC gene exons to identify distinct missense mutations in two HCM kindreds. Clinical, ECG, and echocardiographic studies were performed in the two kindreds: kindred 2755 with amino acid 908Leu→Val mutation and kindred 2002 with amino acid 403Arg→Gln mutation. The morphological appearances of HCM were similar in these two kindreds. However, the two kindreds differed with respect to disease penetrance, age of onset of disease, and incidence of premature sudden death. Twelve of 31 adults (≥ 17 years) with the disease gene in kindred 2755 did not have left ventricular hypertrophy (LVH), and only five of these had ECG abnormalities. Thus, the disease penetrance in adults with this mutation was only 61%. None of 11 children aged < 16 years had LVH. The 908 mutation was associated with a low incidence of cardiac events: Only two sudden deaths and one syncope occurred in 46 individuals with the mutant allele. In contrast, LVH was present in all 11 adults in kindred 2002 with the 403 mutation (100% disease penetrance). In addition, three of four affected children were symptomatic and had clinical evidence of HCM. The disease in this kindred was severe and resulted in six premature sudden deaths. Seven additional patients had syncope or presyncope. ConclusionsIn some kindreds, the HCM disease gene is more prevalent than indicated by echocardiography and ECG. Some point mutations may be associated with a more malignant prognosis. Preclinical identification of children with mutations associated with a high incidence of sudden death and syncope provides the opportunity to evaluate efficacy of early therapeutic interventions.

Abnormal contractile properties of muscle fibers expressing beta-myosin heavy chain gene mutations in patients with hypertrophic cardiomyopathy.

Missense mutations in the beta-myosin heavy chain (beta-MHC) gene cause hypertrophic cardiomyopathy (HCM). As normal and mutant beta-MHCs are expressed in slow-twitch skeletal muscle of HCM patients, we compared the contractile properties of single slow-twitch muscle fibers from patients with three distinct beta-MHC gene mutations and normal controls. Fibers with the 741Gly-->Arg mutation (near the binding site of essential light chain) demonstrated decreased maximum velocity of shortening (39% of normal) and decreased isometric force generation (42% of normal). Fibers with the 403Arg-->Gln mutation (at the actin interface of myosin) showed lowered force/stiffness ratio (56% of normal) and depressed velocity of shortening (50% of normal). Both the 741Gly-->Arg and 403Arg-->Gln mutation-containing fibers displayed abnormal force-velocity relationships and reduced power output. Fibers with the 256Gly-->Glu mutation (end of ATP-binding pocket) had contractile properties that were indistinguishable from normal. Thus there is variability in the nature and extent of functional impairments in skeletal fibers containing different beta-MHC gene mutations, which may correlate with the severity and penetrance of the disease that results from each mutation. These functional alterations likely constitute the primary stimulus for the cardiac hypertrophy that is characteristic of this disease.

Adult murine skeletal muscle contains cells that can differentiate into beating cardiomyocytes in vitro.

It has long been held as scientific fact that soon after birth, cardiomyocytes cease dividing, thus explaining the limited restoration of cardiac function after a heart attack. Recent demonstrations of cardiac myocyte differentiation observed in vitro or after in vivo transplantation of adult stem cells from blood, fat, skeletal muscle, or heart have challenged this view. Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion. We now show a novel population of nonsatellite cells in adult murine skeletal muscle that progress under standard primary cell-culture conditions to autonomously beating cardiomyocytes. Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials. Within 2 d after tail-vein injection of these marked cells into a mouse model of acute infarction, the marked cells are visible in the heart. By 6 d they begin to differentiate without fusing to recipient cardiac cells. Three months later, the tagged cells are visible as striated heart muscle restricted to the region of the cardiac infarct.

The in vitro motility activity of β-cardiac myosin depends on the nature of the β-myosin heavy chain gene mutation in hypertrophic cardiomyopathy

Several mutations in the β-myosin heavy chain gene cause hypertrophic cardiomyopathy. This study investigates (1) the in vitro velocities of translocation of fluorescently-labelled actin by β-myosin purified from soleus muscle of 30 hypertrophic cardiomyopathy patients with seven distinct β-myosin heavy chain gene mutations: Thr124Ile, Tyr162Cys, Gly256Glu, Arg403Gln, Val606Met, Arg870His, and Leu908Val mutations; and (2) motility activity of β-myosin purified from cardiac and soleus muscle biopsies in the same patients. The velocity of translocation of actin by β-myosin purified from soleus or cardiac muscle of 22 normal controls was 0.48 ± 0.09 μm s−1. By comparison, the motility activity was reduced in all 30 patients with β-myosin heavy chain gene mutations (range, 0.112 ± 0.041 to 0.292 ± 0.066 μm s−1). Notably, the Tyr162Cys and Arg403Gln mutations demonstrated significantly lower actin sliding velocities: 0.123 ± 0.044, and 0.112 ± 0.041 μm s−1, respectively. β-myosin purified from soleus muscle from four patients with the Arg403Gln mutation had a similar actomyosin motility activity compared to β-myosin purified from their cardiac biopsies (0.127 ± 0.045 μm s−1 versus 0.119 ± 0.068 μm s−1, respectively). Since these seven mutations lie in several distinct functional domains, it is likely that the mechanisms of their inhibitions of motility are different

Sensing Stretch Is Fundamental

Stretch induces changes in cardiomyocyte biology that are implicated in heart failure, but the mechanism by which stretch is sensed and signals are transduced is unknown. New understanding of the Z disc elements of contractile units are beginning to elucidate the mechanism of stretch sensing and its relation to cardiac adaptation and disease.

Relationship of the C242T p22phox gene polymorphism to angiographic coronary artery disease and endothelial function

Patients with coronary artery disease (CAD) have impaired endothelial function in part due to an increase in vascular oxidant stress. p22phox, an essential component of the NADPH oxidase, is thought to play a critical role in the generation of superoxide anions in the vessel wall. The C242T polymorphism, located in the potential heme-binding site of the p22phox gene, has recently been reported to confer a protective effect on CAD risk in a Japanese study population. In a U.S. population of 252 patients (83% Caucasian) undergoing angiography for diagnosis of CAD, we investigated whether the C242T polymorphism was associated with the presence of CAD. In a subset of 142 patients, we studied whether the polymorphism manifests its potential protective effects through alteration of vascular endothelial function by measuring coronary epicardial and microvascular responses to intracoronary acetylcholine and sodium nitroprusside. Prevalence of the C242T allele was not different in 149 patients with CAD compared to 103 patients with angiographically normal coronary arteries (65.1% vs. 54.4%, P = 0.11). The C242T allele frequency in our population was nearly fourfold higher than reported previously in a Japanese population. There were also no significant differences in coronary epicardial or microvascular responses to acetylcholine or sodium nitroprusside between groups of patients with or without the C242T allele. In a U.S. population, the C242T polymorphism does not appear to confer protection from endothelial dysfunction or CAD. Am. J. Med. Genet. 86:57-61, 1999. Published 1999 Wiley-Liss, Inc.